The goal of this proposal is to understand the structure and assembly of the vaccinia virion core. The core contains the viral DNA and at least 50 viral proteins. Core substructures can be distinguished by electron microscopy, including lateral bodies, a core wall, and a tubular structure in the lumen of the core, however very little is known about the precise protein composition of each substructure, the function of the individual proteins within the core, or the role of individual core proteins in core assembly during virus morphogenesis. The project will focus on a subset of nine core proteins that break down into four categories: 1) a cysteine protease required for cleavage of the major structural virion proteins (I7L), 2) four putative core wall proteins (A3, A4, A10, F17), 3) two putative tube proteins (H4, L4), and two putative core lumenal proteins (E6 and E8). In one aim the assembly of the pox virion core will be investigated by performing infections with wild type virus or virus bearing mutations in specific core structural proteins, and analyzing the infections by electron microscopy and immunofluorescence confocal microscopy. Infections will be monitored not only for the specific structures formed but also for the distribution within virus factories of each of the proteins and the viral DNA. These studies will reveal how individual components of the core are trafficked during morphogenesis, and how each component contributes to the evolution of the final core structure. In a second aim the structure of the pox virion core will be investigated by performing controlled degradation of wild type virions or virions bearing mutations in specific core structural proteins, and analyzing the virions and their degradation products by electron microscopy, gel electrophoresis and western blotting. Virion substructures will be probed with a panel of antibodies specific for proteins hypothetically contained in each of the major virion substructures, including lateral bodies, core wall, tube and lumen. Analysis of wild type virions in this fashion will reveal the localization of specific proteins within specific core substructures. Analysis of the mutant virions will reveal defects in specific core substructures, thus reinforcing assignments made from studies of wild type virions, and providing information on the function of each protein within the core structure.